The decomposition of chemiluminescent chemical compounds to release electromagnetic, and especially optically detectable, energy--usually luminescence in the form of visible light--is well known and understood. The incorporation of such light emitting reactants in art-recognized immunoassays, chemical assays, nucleic acid probe assays, and physical and chemical probe techniques as a means by which the analyte, a substance whose presence, amount or structure is to be determined, is actually identified or quantified, has assumed increasing importance in recent years, particularly with the advent of enzymatically-cleavable water-soluble 1,2-dioxetanes. See, for example, copending Bronstein U.S. Pat. Ser. No. 889,823, "Method of Detecting A Substance Using Enzymatically-Induced Decomposition of Dioxetanes", filed Jul. 24, 1986; Bronstein et al. U.S. patent application Ser. No. 140,035, "Dioxetanes For Use In Assays", filed Dec. 31, 1987; Edwards et al. U.S. patent application Ser. No. 140,197, "Synthesis of 1,2-Dioxetanes and Intermediates Therefore", filed Dec. 31, 1987; and Voyta et al. U.S. patent application on "Chemiluminescence Enhancement", filed Jun. 1, 1988, U.S. Ser. No. 286,725.
When these chemiluminescent 1,2-dioxetane derivatives are used to analyze and study substances in biological systems, and as biological systems are aqueous, the chemiluminescent compound or compounds used should be soluble in a polar protic environment, especially in aqueous media. Thus, the present invention particularly relates to the purification of chemiluminescent 1,2-dioxetane derivatives that 1) can be induced to decompose to yield a moiety in an excited state, such moiety having a heteropolar character that makes it susceptible to environmental effects, and that 2) are usable to determine the presence, concentration or structure of a substance in a polar protic environment, particularly a substance in an aqueous sample.
The prior art methods for purification of dioxetanes of which applicants are aware were designed for dioxetanes that are soluble only in organic solvents. Thus, Adam et al., J. Org. Chem. 49:3920 (1984), carried out the synthesis of aryl-substituted 1,2-dioxetanes in methylene chloride, evaporated the solvent at reduced pressure, chromatographed the residue on silica gel in a mobile phase consisting of 4:1 petroleum ether:methylene chloride at low temperatures, then recrystallized the product from organic solvents. Schaap et al., Tetrahedron Letters, 28:935 (1987), carried out the synthesis of adamantyl methoxy naphthyl 1,2-dioxetane in methylene chloride, evaporated the solvent in vacuo, and then recrystallized the dioxetane from pentane/ether. Hummelen et al., Methods In Enzymology, 133:531 (1986), synthesized a series of compounds based on adamantylidene adamantane 1,2-dioxetane in organic solvents and, after removal of the solvents in vacuo, used the compounds without further purification. Also using dioxetane products without further purification were Schaap et al., J. Amer. Chem, Soc., 104:3504 (1982), who carried out the synthesis of phenol-substituted 1,2-dioxetanes in acetone solvent and then simply evaporated the solvent in vacuo to give an oily product. Schaap in published European Patent Application No. EP 87108978 discloses purifying water-insoluble derivatives of 1,2-dioxetanes by preparative thin layer chromatography in organic solvents, and by recrystallization at -25.degree. C. in hexane or pentane. Although a water-soluble dioxetane, namely, a phosphorylated xanthone derivative, is discussed in this published European application, this compound was synthesized and used without any reported purification. Baumstark et al., J. Org. Chem. 48:3713 (1983), purified 3-methyl-3-ethyl-1,2-dioxetanes in carbon tetrachloride on a silica gel column in pentane at -30.degree. C.
Water-soluble arylphosphate compounds other than phosphorylated 1,2-dioxetanes or analogous enzymatically, chemically or thermally clearable chemiluminescent compounds have been purified by recrystallization. For example, 4-methylumbelliferyl phosphate was recrystallized from ethanol/diethyl ether in low yield (Fernlay, M., et al. Biochem. J. 97:95 (1965)). Although barium salts or salts with organic bases such as cyclohexylamine are often used to enhance the crystallinity of organic phosphate molecules (Reese, C. B., et al., J. Chem. Soc. (C) 2092 (1970)), dioxetanes, whether phosphorylated or not, and amines are incompatible [Lee, D. C-S. et al. in "Chemiluminescence and Bioluminescence", eds. Cormier, M. J. et al. (New York: Plenum Publishing Corp., 1973,) p. 265)]. (-)-5-Enolpyruvylshikimate-3-phosphate.Na.sub.4 + was successfully purified by ion exchange chromatography using a triethylammonium bicarbonate buffer gradient (Chouinard, et al. J. Org. Chem. 51:76 (1986)). As noted above, however, dioxetanes, being sensitive to amine-catalyzed degradation are unstable in the presence of an amine base (see Examples below).
The marked instability of dioxetanes in low pH media has been noted repeatedly (see, for example, Bartlett, P. D., Chem. Soc. Reviews 5:149 (1976)). Acidity produced by solid phases, e.g., silica gel, and the low pK.sub.a of alcoholic solvents are also known to decompose dioxetanes (see, for example, Zaklika, K. A. Photochem, Photobiol. 30:35 (1979), and McCapra, F., et al., JCS Chem. Commun. 944 and 946 (1977)).
The destructive effect of acidity or amines on dioxetanes means, unfortunately, that the most useful of the buffer salts customarily used in liquid chromatography--ammonium salts (i.e., ammonium acetate or ammonium carbonate)--cannot be used to purify water soluble dioxetanes by this method. In traditional purification schemes in which such salts can be used, they will customarily be added in amounts sufficient to put them in equilibrium with amine and acid components which are volatile in high vacuum, even at the low temperatures at which such procedures are usually carried out. This is important as it provides a simple means of removing these buffer salts during the isolation of water-soluble compounds: removal of the solvent and the buffer salt in vacuo. If, however, one attempts to use ammonium salts as buffers when purifying dioxetanes the acidic moiety produced during evaporation produces a pH that will, as the concentration of the acidic moiety increases, in turn decompose the water soluble dioxetane one is trying to purify.
High performance liquid chromatography (HPLC), also known as high pressure liquid chromatography, has been used to study the thermal stability of several water-insoluble tricyclodecane spiro phenoxy 1,2-dioxetanes following purification of these compounds by recrystallization from hexane at -78.degree. C. [Jefford, C. W., et al. J. Chromat. 347:183-7 (1985)]. Although the dioxetanes did not decompose during HPLC on a LiChrosorb Si60 column, it is important to note that, because of the water-insolubility of the dioxetanes separated, HPLC had to be performed with organic solvents, that is, the dioxetanes were injected onto the column in o-xylene solution, and isoctane-tetrahydrofuran mixtures were used as the mobile phase. Such systems cannot be used to purify water-soluble chemiluminescent 1,2-dioxetane derivatives such as those used in aqueous analytical systems.
In silica columns for liquid chromatography, at pH values at or above 7.5 where dioxetanes are stable, the silica packing is unstable and dissolves, as shown by the data taken from the Waters Associates, Inc. Manual of Liquid Chromatography (1988). Contrariwise, at acidic pH values, at which silica columns are most stable, dioxetanes are unstable (Zaklika et al. supra).
To sum up, then, the purification of water-soluble dioxetanes cannot be accomplished satisfactorily (i.e., without decomposing the dioxetane one is attempting to purify) in low pH media, whether buffered or not, using either conventional MPLC or medium pressure liquid chromatography (MPLC) systems in which a silica- or crosslinked polymer-based reversed phase adsorbent is used as the stationary phase and a hydrophobic organic solvent or solvent system is used as the mobile phase, or conventional low pressure liquid chromatography (LPLC) systems.